Background: A temporarily elevated level of serum mast cell tryptase (ST) indicates mast cell activation and occurs in systemic anaphylactic reactions (SAR). We measured ST following a sting challenge in vespid venom-allergic patients treated with venom immunotherapy (VIT) and in healthy controls, respectively. Aim of the study: To assess changes of ST over time in vespid venom-allergic patients at the occasion of a re-sting and in healthy controls. Methods: A sting challenge was performed in 20 patients on vespid VIT to monitor efficacy of VIT. ST was measured between 9.00 and 10.00 a.m. (baseline). Sting challenge was performed at 2.00 p.m., and ST was determined again 20 min, 90 min and 18 h later. Measurements at corresponding times of the day were done in nine healthy controls. Results: One patient developed a mild SAR to the sting challenge which was associated with a temporary increase of ST. In the other 19 patients who tolerated the sting challenge without SAR ST decreased significantly by 18.0% (median, range 8.3–36.7%). Twenty minutes after the sting when compared with baseline levels (P P P = 0.57). A comparably significant temporary decline was found in controls. Conclusions: The temporary decline of ST in patients and in controls suggests a circadian variation of ST concentration. A normal diurnal pattern of ST concentration after sting challenge is associated with successful treatment.